Constitutive production of interleukin 2 by a T cell hybridoma

ABSTRACT

A process for constitutively producing murine IL-2 from hybridoma cells generated by fusing nitrogen stimulated malignant murine cells with drug sensitive murine thymoma driver cells. A fusing agent is used to fuse the two parent cells together. After fusion, the hybrid cells are cultured in vitro in a supplemented, serum containing tissue culture medium to thereby constitutively produce IL-2. The medium also includes a group of suppressing compounds which will prevent unfused driver cells from replicating, and feeder cells used to nurture the growth of competent hybrid cells.

DESCRIPTION

1. Technical Field

The present invention relates to a process of preparing interleukin 2(hereafter "IL-2") (formerly known in literature as "T Cell growthfactor" or "TCGF"), and more particularly to a process forconstitutively producing murine II-2 from hybrid cells formed by thefusion of murine lymphoma cells with murine IL-2 producing cells.

2. Background Art

IL-2 is a soluble protein which is capable of influencing cell-mediatedimmune responses in mammals, including: (1) enhancement of thymocytemitogenesis; (2) production of alloantigen-directed cytotoxic Tlymphocytes in thymocyte and nude spleen cell cultures; and (3)assistance in the generation of helper T cells in antibody responsesfollowing stimulation with hetrologous erythrocytes. In addition, IL-2is capable both of sustaining the in vitro expotential proliferation ofeffector T cell lines and of inducing, both in vitro and in vivo, thegeneration of cytotoxic T cells from nude mouse spleens.

In the past, murine IL-2 has been produced by culturing normal rat andmouse spleen cells in tissue culture medium and stimulating the cellswith a plant mitogen, such as phytohemagglutinin (hereafter "PHA") orconcanavalin A (hereinafter "Con A"). However, producing murine IL-2 bymitogen stimulation of normal spleen cells results in weekconcentrations of Il-2. Very large volumes of IL-2 containingconditioned medium must be fractionated to produce only small quantitiesof purified murine IL-2. As a consequence, sufficient quantities ofconcentrated murine IL-2 have not been available for experimentation,nor for effective study of the molecular characterization of thisimmunoregulatory molecule.

Gillis et al. have recently documented a process for producing murineIL-2 from tumor cells by stimulation of the cells with a T cell mitogen."Biochemical and Biological Characterization of Lymphocyte RegulatoryMolecules-III. The Isolation and Phenotypic Characterization ofInterleukin 2 Producing T Cell Lymphomas", 125 The Journal of Immunology2570 (1980). A particular murine splenic lymphoma cell line from theB10. BR mouse, designated as LBRM-33, was found to produce severalhundred times more IL-2 per milliliter than previously generated bymitogen stimulation of identical numbers of normal mouse splenocytes.Gillis et al., supra, also reported producing IL-2 from cloned LBRM-33cells by culturing the cloned cells in tissue culture medium andstimulating the cells with a plant mitogen. Several of the cloned celllines were found to produce higher concentrations of IL-2 than generatedby identical numbers of the parent LBRM-33 cells.

However, in conventional procedures for producing IL-2, the T cellmitogen remains in the IL-2 containing supernate generated by mitogenstimulation of the murine cells. In the process of recovering the IL-2from the supernate, it is important that all of the mitogen is removed.For instance, when IL-2 is used to initiate T cell line proliferation,even trace amounts of T cell mitogen can activate T cell subpopulationswhich will respond to IL-2 and replicate. This may result in theexpression of spurious immune reactivities and specificities. Althoughthe mitogen can be removed from the supernate, the procedure is timeconsuming and complicated, requiring many separate biochemical steps.Another drawback of producing IL-2 by stimulating LBRM cloned cells witha plant mitogen is that within a short time after the IL-2 containingsupernate is elaborated, the productive cell line dies, thus requiring acontinuous supply of productive cells to generate significant quantitiesof IL-2. Accordingly, a principal object of the present invention is toconstitutively, i.e. without the need of a T-cell mitogen as astimulant, produce murine IL-2 from a cell line which continues to liveafter elaborating IL-2 containing supernate.

Schrader et al. reported producing factors affecting haematopoieticcolony-forming cells and B cell antibody responses from hybrid cellsresulting from the fusion of murine T lymphoma cells, designated as "BW5147", (obtained from a leukemic BALB/c mouse) with blast cells isolatedfrom lymph nodes of mice immunized with keyhole limpet haemocyanin.However, the T cell mitogen, Con A, was required to be added to theculture medium to produce the factors. "A Con A-Stimulated T-CellHybridoma Releases Factors Affecting Haematopoietic Colony-Forming Cellsand B-Cell Antibody Responses", 283 Nature 197 (1980). Harwell et al. in"A Concanavalin A Inducible, Interleukin 2 Producing T Cell Hybridoma",152 Journal of Experimental Medicine 1893 (1980), discussed producing ahybrid cell line having T cell characteristics. The hybrid cells wereformed by fusing the mouse T lymphocyte cell line, BW-5147, with Con Astimulated spleen cells. However, liberation of IL-2 activity intotissue culture supernate also required mitogen stimulation of the hybridT cells.

DISCLOSURE OF THE INVENTION

The present invention relates to a process for constitutively producingIL-2 from a T cell hybridoma. The process includes first fusing IL-2producing malignant murine cells with murine drug-sensitive driver cellsand then culturing the resulting hybrid T cells in a medium withoutrequiring stimulation of the hybrid T cells with a T cell mitogen. Priorto the fusion process, malignant murine IL-2 producer cells aresuspended in a culture medium and stimulated with a T cell mitogen. Alsoprior to hybridization, murine neoplastic cells are drug-sensitized toform driver cells. The stimulated IL-2 producing malignant murine cellsare mixed with the drug-sensitized driver cells and then a fusing agentadded thereto to produce hybrid T cells which exhibit both the IL-2generating capacity of the malignant murine producer cells and theimmortality of the drug-sensitized driver cells.

IL-2 is constitutively produced by culturing the hybrid cells, in vitro,in a protein containing medium supplemented with various additives. Aseries of selective suppressor drugs are added to the medium to killresidual unfused driver cells. Prior mitogen stimulation of malignantmurine IL-2 producer cells, insures that an unfused producer cell willdie. Furthermore, feeder cells (thymocytes) are also added to the hybridT cells to ostensibly provide them with nutrients. In the culturingprocess, IL-2 is continuously produced without the presence of amitogen, and moreover, the hybrid cells continue to live as long astissue culture medium is present. Another advantage of producing IL-2 inthis manner is that the supernate generated in the culture does not haveto be treated to remove contaminating T cell mitogen.

The process of the present invention also includes cloning murine hybridT cell lines by suspending single cell samples of hybrid T cells foundconstitutively to produce IL-2 in vitro. After the single cell cultureshave grown to appropriate densities, the cells are resuspended in aculture medium to generate IL-2 continuously without having to stimulatethe hybrid T cells with a mitogen.

The above-described hybridizing process has been used in conjunctionwith a particular murine radiation-induced splenic lymphoma cell linewith the B10. BR mouse, designated as LBRM-33 together with a particularmurine T-lymphoma, drug-sensitized driver cell line designated as BW5147. The IL-2 producing portion of the process of the present inventionhas been carried out with hybrid T cells generated by fusion of thesetwo cell lines and by clones of such hybrid T cells. Applicant hasidentified a particular clonal hybrid cell line, designated asLBRM-33-1A8C3C10 (hereafter "1A8C3C10"), which is capable ofconstitutively producing IL-2 in concentrations approximately ten timesgreater than that produced by equivalent numbers of mitogen stimulatednormal mouse spleen cells.

Applicant has established that an initial cell concentration of theparticular hybrid T cell line used in the process of the presentinvention affects the level of IL-2 produced. Also, the quantity of IL-2produced appears to be directly related to the time that the hybrid Tcells are cultured in an appropriate culture medium.

Brief Description of the Drawings

The details of typical embodiments of the present invention aredescribed in connection with the accompanying drawings in which:

FIG. 1 is a graph illustrating the screening process used for theselection and ultimate cloning of the IL-2 producer hybridoma cell line,1A8C3C10, with cloning trials progressing from the upper panel(identification of the 1A8 hybrid) to subcloning of the 1A8C3C10producer hybrid cells (lowest panel);

FIG. 2 is a graph illustrating units of IL-2 activity per milliliter forvarious initial cell concentrations of the 1A8C3C10 cell line that werecultured for various numbers of days in Click's medium supplemented withten percent (10%) by volume FSC;

FIG. 3 is a graph illustrating growth curves of CTLL cells cultured inthe presence of 1A8C3C10 hybridoma cell conditioned medium and ConA-stimulated rat spleen cell conditioned medium, both adjusted to finalIL-2 activity level of 2.5 units per milliliter prior to use in the CTLLgrowth cultures; and,

FIG. 4 is a graph showing the ability of three different log₂ dilutionseries of IL-2 conditioned media to test the ability of such conditionedmedia to augment mitogenesis of CBA/J6 female mice thymocytes (2×10⁶cells per milliliter), with the three different cultures composed of:(1) 1A8C3C10 hybridoma supernate adjusted to a concentration of 10 unitsof IL-2 per milliliter; (2) supernate from cultures of rat spleen cellsstimulated for 24 hours with Con A (5 micrograms/milliliter) andadjusted to a concentration of 30 units of IL-2 per milliliter; and (3)LDRM-33 cells stimulated by one percent (1%) by volume PHA and adjustedto a concentration of 100 units of IL-2 per milliliter.

BEST MODE OF THE INVENTION Outline of Process

In accordance with the present invention, murine malignant neoplasticcells are cultured in vitro in a protein containing medium andstimulated with a T cell mitogen. The mitogen stimulated cells are fusedwith drug-sensitive murine lymphoma driver cells by mixing the twodifferent cells together and then adding a fusing agent. Thereafter, themixture is pelleted, washed in a protein containing medium and the cellsolution pelleted again. The resultant hybrid T cell containing pelletis next resuspended in a protein containing medium which is supplementedwith various additives, feeder cells and a series of suppressorchemicals which will cause the unfused driver cells to die.

The present invention also concerns identifying potent constitutive IL-2producing cell lines by cloning hybrid T cell lines found to produceIL-2 without requiring mitogen stimulation. Cloning is accomplished by alimiting dilution procedure wherein constitutive IL-2 producer hybridcell lines are cultured in a supplemented, protein containing medium ata cell density of one cell per milliliter. After a period of time, thecells which produce the highest titers of IL-2 are subcloned, again by alimiting dilution method, to seek cell line sources capable of evengreater IL-2 production. Once isolated, such clonal cell lines arecultured in a supplemented medium to produce IL-2 without the presenceof a T cell mitogen.

The processes of the present invention have been applied to aradiation-induced splenic lymphoma cell line, designated as LBRM-33,derived from the B10.BR mouse. This murine lymphoma cell line has beenfused with a hypoxanthine-aminopterin-thymidine drug (hereafter"HAT")-sensitive murine T-lymphoma cell line, designated as BW 5147, toproduce several hybrid cell lines, see FIG. 1 below, capable ofconstitutive IL-2 production. Cloning of one of the most potent hybridcell lines, designated as LBRM-33-1A8 (hereafter "1A8"), has resulted inthe identification of various clonal hybrid T cell lines which arecapable of constitutively producing IL-2 including a cell linedesignated as LBRM-33-1A8C3 (hereafter "1A8C3"). An even more potentIL-2 producing cell line, designated as LBRM-33-1A8C3C10 (hereafter"1A8C3C10") has been isolated by subcloning the 1A8C3 clonal cell line.A microassay of the activity level of IL-2 constitutively produced bythe 1A8C3C10 cell line by use of the microassay procedure discussed byGillis et al in "T-cell growth Factor: Parameters of Production and aQuantative Microassay for Activity", The Journal of Immunology 2027(1978), has established that the elaborated supernate stimulated an IL-2dependent T lymphocyte line (hereafter "CTLL") to incorporate in excessof 25,000 counts per minute of tritiated thymidine (hereafter "[³H]Tdr]") at a final dilution of 1:10. This activity level is more thanten times the activity level of IL-2 produced by mitogen stimulation ofequivalent members of normal mouse splenocytes. The results of themicroassay of the supernates generated by the parent hybrid T cell line1A8 and its clones and subclones are set forth in FIG. 1.

As outlined above, in the IL-2 production process of the presentinvention, prior to fusion, the IL-2 producer cells, such as the LBRM-33lymphoma cells, are initially stimulated with a T cell mitogen. Thesemitogens may include various commercially available standardglycoproteins, such as PHA, Con A and pokeweed mitogen (hereafter"PKM"). Although different concentrations of a particular mitogen may beemployed, applicant has found that if PHA is used, a concentration ofapproximately 1% by volume is adequate to stimulate the LBRM-33 lymphomacells into IL-2 production.

The process of the present invention also involves several culturingprocedures, including during: (i) initial stimulation of the parent IL-2producer cell line with a mitogen prior to hybridization; (ii) fusion ofthe IL-2 producer cells and the drug-sensitive driver lymphoma cells;(iii) cloning and subcloning of the hybridoma T-cells; and (iv)constitutive IL-2 production by parent and clonal hybrid T cells, asdiscussed above. Various types of appropriate cell culturing media,which have been previously found to foster growth of T lymphocytes, maybe utilized in these different culturing steps. These culture mediainclude Roswell Park Memorial Institute medium 1640 (hereafter "RPMI"),Click's medium and Dulbecco Modified Eagle's medium (hereafter "DMEM").

In the production of IL-2 containing supernates from the parent andclonal hybridoma cells, the culture media may be supplemented withvarious individual additives, or combinations of additives, includingfetal calf serum (hereafter "FCS") which has been heat-inactivated by,for example, applying heat at 56° C. for approximately 30 minutes. Thequantity of FCS added may be from 2 to 10% of the total culture volume.Another additive is penicillin at a concentration range of approximately25-250 units per milliliter, and preferably approximately 50 units permilliliter. Streptomycin also may be utilized as an additive in apreferred concentration range of from 20-250 micrograms per milliliterof total culture volume, and ideally approximately 50 micrograms permilliliter. Further additives include: (i) sodium pyruvate in aconcentration range of approximately 10 to 150 millimolar and ideallyapproximately 100 millimolar; (ii) N-2-hydroxy-piperazine-XI¹-2-ethene-sulfonic acid (hereafter "HEPE'S") buffer in a preferredconcentration of from 10 to 60 millimolar, and ideally approximately 25millimolar; and (iii) fresh L-glutamine in a preferred concentrationrange of approximately 150-500 micrograms per milliliter, with an idealconcentration of approximately 300 micrograms per milliliter. Inaddition, NaHCO₃ at a concentration range of 1 to 30 millimolar andideally about 16 millimolar may be added to the culture media.

The above discussed culturing processes for: (i) stimulation of anoriginal murine malignant cell line, specifically LBRM-33 lymphomacells; (ii) fusion of the stimulated LBRM-33 lymphoma cells withdrug-marked driver lymphoma cells; and (iii) constitutive production ofIL-2 from hybrid and clonal hybrid T cell lines may be carried out invarious environmental conditions. Preferably, however, the culturesshould be maintained at a temperature range of approximately 35° to 38°C. and in a humidified atmosphere of approximately 5 to 10% CO₂ in air.Also, ideally the pH of the culture medium should be kept in slightlyalkaline condition, in the range of approximately pH 7.2 to 7.4.

Different types of murine driver cells may be fused with the lectinstimulated malignant murine cells to impart the hybrid T cells with theability to proliferate after fusion. One such cell line is the mouse Tlymphoma cell line, known as BW 5147, derived from the BALB/c mouse.Other driver cell lines include any T cell lymphoma selected to be8-azoguanine/ouabain resistant. Lymphoma cells selected to be resistantto azoguanine or ouabains, are most likely sensitive to HAT, and willdie when cultured in tissue culture medium containing HAT.

According to the present invention, the driver cell line is drug-markedor sensitized by conventional techniques to prevent reproduction of thedriver cells. Thereafter when used in cell fusion experiments,subsequent culture of hybrid cells in tissue culture medium containingHAT, prevents unfused driver cells from proliferating. Various drugswhich may be used to sensitize driver cells including the BW 5147 Tlymphoma cells, include ouabain, glutathione and 8-azoguanine.

The fusion step of the present invention involves combiningdrug-sensitive driver cells with lectin stimulated IL-2 producing cellsby placing such cells in a slightly alkaline tissue culture medium andthen gradually adding a fusing agent. Fusing agents may include varioustypes of condensation polymers of ethylene oxide and water, such aspolyethylene glycol (hereafter "PEG"). Other fusing agents include DNAtransforming viruses, such as Sendai viruses or the fusion proteinobtained therefrom. For optimum fashion, the quantity and concentrationof the fusing agent must be controlled. For instance, if PEG is used tofust lectin stimulated LBRM-33 lymphoma cells with HAT-sensitive BW 5147cells, approximately 1 milliliter of 40% PEG (weight/volume) should beadded to the tissue culture medium. However the volume of PEG may rangefrom 0.5 to 3 milliliters and the concentrations of PEG may vary from35% to 60% (weight/volume).

During the process of culturing the fused cells and the clones thereof,feeder cells may be added to the culture medium both to induce theproliferation of the fused cells. Although not yet definitely confirmed,it is considered that the feeder cells function to provide optimal celldensity to allow small numbers of hybrid cells to multiply more readily.Also, it is thought that the feeder cells may provide the hybrid cellswith nutrients required for their proliferation. Various types of feedercells may be employed, including thymocytes from the BALB/c mouse. Othertypes of feeder cells include murine spleen cells, irradiated murineperitoneal exudate cells and murine macrophage cells. Feeder cellsshould be added to HAT containing cultures at concentrations rangingfrom 0.5 to 5.0×10⁶ cells/milliliter with an optimal concentration ofabout 3×10⁶ cells/milliliter.

The present invention also includes identifying potent sources of IL-2by cloning hybrid T cell lines known constitutively to producesignificant quantities of IL-2. Thereafter, the cloned hybrid T celllines are cultured in a medium supplemented with various additives insubstantially the same manner in which IL-2 is constitutively producedfrom hybrid 1A8 cells, as outlined above. Cloning is accomplished by alimiting dilution procedure wherein cells from parent hybrid cell lines,such as 1A8 cells, are cultured in flatbottom microplate wells. The cellare individually seeded in 100 microliter volumes of Click's mediumsupplemented with a 10% by volume quantity of FCS. Feeder cells, such asthymocytes from BALB/c mouse, are added to the culture medium to enhancecell growth. After approximately eight days in culture, supernates fromthe microplate wells which house viable cell growth are harvested andtested for IL-2 -activity.

Clonal cell lines found to produce the highest titers of IL-2, such asthe hybrid cell line designated as 1A8C3, are then subcloned to seekeven more potent IL-2 producing cell lines. The technique followed forsubcloning is the same as used during the original cloning process, asdescribed in the above paragraph. Use of this procedure to subclone thehybrid 1A8C3 cells has led to the identification of an even more potentIL-2 producing cell line, designated as 1A8C3C10. As illustrated in FIG.1, the 1A8C3C10 cell line was found to produce even greater quantitiesof IL-2 than generated by the same number of 1A8C3 cells as indicated bya higher level of [³ H]Tdr incorporation.

Applicant has found that the initial cell density of a particularhybridoma cell line used in the culture process affects the quantity ofIL-2 constitutively produced per number of initial cells. For instance,as set forth in FIG. 2, using the 1A8C3C10 hybrid cell line, when aconcentration of 1×10⁵ 1A8C3C10 cells per milliliter are cultured withClick's medium supplemented with 10% by volume FCS in 10 millilitervolumes (25 square centimeter tissue culture flasks, #3013, FalconPlastics. Oxnard, CA), approximately 6 units of IL-2 activity permilliliter are produced after four days of culture. When the initialcell concentration is increased to approximately 5×10⁵ cells permilliliter, a maximum of approximately 10 units of IL-2 per milliliterare produced. If, however, the concentration of 1A8C3C10 cells isfurther increased to, for instance 1×10⁶ cells per milliliter, theproduction of IL-2 drops back down to approximately 4 units permilliliter. Thus, if 1A8C3C10 cells are used to produce IL-2, theinitial cell density preferably should be in the range of about 3×10⁵cells per milliliter to 7×10⁵ cells per milliliter with an idealconcentration of approximately 5×10⁵ cells per milliliter.

The quantity of IL-2 constitutively generated by the hybrid murine cellsproduced by the present invention varies with time. For instance, when5×10⁵ 1A8C3C10 cells per milliliter are cultured in Click's mediumsupplemented with 10% by volume FCS, approximately 3 units permilliliter of IL-2 activity are generated after twenty-four hours ofculturing, see FIG. 2. At three days, IL-2 activity increases toapproximately 5 units per milliliter. A peak level of approximately 10units per milliliter of IL-2 activity is obtained after four days ofculturing. Thus, the optimal culture duration for constitutivelyproducing IL-2 from 1A8C3C10 clonal hybrid T cells is approximately fromthree to five days.

Microassay of IL-2

The level of IL-2 produced by the hybridoma culture supernates of thepresent and clonal hybrid cells of the present invention (to ascertain,for example, what cell lines produce significant levels of IL-2), may betested by using the microassay procedure discussed by Gillis et al in"T-Cell Growth Factor: Parameters or Production and a QuantatativeMicroassay for Activity", 120 The Journal of Immunology, 2027 (1978).The assay monitors the IL-2 dependent cellular proliferation of a mousecytotoxic T cell line (hereafter "CTLL"). Once potent hybrid cell linesare identified, such as 1A8C3C10 cells, the microassay technique is thenemployed to determine optimum culture conditions for IL-2 production,such as optimum initial cell concentrations of 1A8C3C10 and optimumharvest times, as discussed above.

Briefly, the microassay procedure includes seeding approximately 4×10⁴CTLL cells in a log₂ dilution series of potential IL-2-containing hybridsupernate samples. In each culture, approximately 4×10⁴ CTLL cells aresuspended in Click's medium supplemented with 10% by volume FCS tothereby form 200 microliter volumes. The cultures are incubated forapproximately 20 hours at 37° C. in a humidified atmosphere of 5% carbondioxide in air. Thereafter, the cultures are pulsed for approximatelyfour hours with 0.5 microcurie of [³ H]Tdr having a specific activity of20 millicures per millimole (obtained from New England Nuclear, Boston,MA). After pulsing, the cultures are harvested onto glass fiber filterstrips, for instance with the aid of a multiple automated sampleharvester. [³ H]Tdr incorporation by the CTLL cells is measured byliquid scintillation counting. By this procedure, the CTLL cells whichare exposed to IL-2 will incorporate [³ H]Tdr in a dose-dependentmanner. CTLL cells cultured in the absence of IL-2 will incorporate onlyscintillant control levels of ³ [H]Tdr and will be more than 95%trypan-blue positive after 24 hours of IL-2 deprivation, indicating thatsuch cells are dead. Units of IL-2 activity are determined by Probitanalysis of [³ H]Tdr incorporation data, as described above. A 1 unitper milliliter standard of IL-2 activity is defined as the amount ofIL-2 activity present in a forty-eight hour tissue culture mediumconditioned by Con A (2.5 micrograms/milliliter) stimulation of aninitial concentration of 10⁶ cells per milliliter of normal rat spleencells, A 1 unit per milliliter standard of IL-2 activity routinelystimulates approximately 2500 counts per minute of CTLL [³ H]Tdrincorporation at a dilution of 1:20. The results of the microassays ofthe supernates produced by the various hybrid T cell lines are set forthin FIG. 1.

Confirmation of the Presence of IL-2 in Hybridoma Culture Supernates

In addition to the above-described microassay procedure, the presence ofIL-2 in the hybridoma culture supernates was further substantiated bytesting their capacity to support long-term growth of CTLL cells. Thistest is of significance, since one of the potential uses of aconstitutive IL-2 producer hybridoma is to generate conditioned mediumwhich is capable of supporting the in vitro growth of cloned effector Tcell lines. In the test procedure, hybrid supernates containing 5 unitsper milliliter of IL-2 activity were dialysed into complete RPMI 1640medium to replenish nutrients lost during IL-2 production by hybrid cellmetabolism. Next, CTLL cultures were prepared by seeding two millilitercultures of CTLL cells in concentrations of 2.5×10³ cells per milliliterin flat-bottomed cluster plate wells (#3024, Costar, Inc., Cambridge,MA). Each of the cultures contained either 50% by volume 1A8C3C10dialyzed conditioned medium or a 50% by volume rat spleen cellconditioned medium to serve as a conrol sample. The rat spleen cellconditioned medium was produced by a concentration of 10⁷ rat spleencells per milliliter stimulated for 24 hours by a concentration of 5micrograms per milliliter of Con A. Both the hybridoma and the ratspleen cell conditioned medium were adjusted to final IL-2 activitylevel of 2.5 units per milliliter prior to use in the CTLL growthcultures. Cell samples from both cultures were harvested daily toascertain their growth rate and their viability was measured by trypanblue dye exclusion. When the CTLL cells reached a density ofapproximately 1×10⁵ cells per milliliter, concentrations of 2.5×10³ ofthe CTLL cells per milliliter were subcultured in fresh 2 millilitertissue culture medium, each containing 25 units per milliliter of IL-2derived from the appropriate source of IL-2 activity, e.g. 1A8C3C10hybrid cell dialyzed conditioned medium or rat spleen cell conditionedmedium.

As illustrated in FIG. 3, both the CTLL cells grown in standard IL-2containing medium or in 1A8C3C10 supernates exhibited identical growthpatterns over a nine day culture. The CTLL cell samples cultured in bothIL-2 sources doubled on both the first and second days of culture andincreased to nearly 10-fold of their original cell density by the thirdday. The correspondence between the proliferation of the CTLL cells inthe two different conditioned media verifies the existence of IL-2activity in 1A8C3C10 supernates. Furthermore, no difference in cellviability was observed between CTLL cells cultured in rat spleen cellconditioned medium or in 1A8C3C10 hybridoma cell conditioned medium, inboth instances more than 90% viability was observed on ach day ofculture.

To further confirm the presence of IL-2 activity, the supernates of thehybrid T cells and of the clones thereof were tested for their capacityto enhance thymocyte mitogenesis, another biologic activity which isattributed to IL-2. The test included seeding concentrations ofapproximately 2×10⁵ cells per milliliter of thymocyte cells preparedfrom CBA/J6 female mice, (8-10 weeks of age, Jackson Laboratories, BarHarbor, Maine) in three different log₂ dilution series of IL-2conditioned media to test the ability of such conditioned media toaugment mitogenesis of the CBA/J6 thymocytes. The three differentcultures included: (1) 1A8C3C10 hybridoma supernate adjusted to aconcentration of 10 units of IL-2 per milliliter; (2) supernate fromcultures of rat spleen cells stimulated for 24 hours with Con A (5micrograms/milliliter) and adjusted to a concentration of 30 units ofIL-2 per milliliter; and (3) LBRM-33 cells stimulated by 1% by volumePHA and adjusted to a concentration of 100 units of IL-2 per milliliter.Each of the thymocyte cultures were 200 microliters in total volume andwere composed of Click's medium, 10% by volume FCS and a concentrationof 2 micrograms per milliliter of Con A. After 72 hours of culture, eachof the cultures was exposed to 0.5 microcuries of [³ H]Tdr as detailedabove in the discussion relating to "Microassay of IL-2". Thereafter,the thymocytes were harvested and cell proliferation measured by liquidscintillation counting. As indicated in FIG. 4, the 1A8C3C10 supernate,the LBRM-33 conditioned medium and the conventionally prepared ratconditioned medium all significantly enhanced Con A induced thymocyteproliferation, thereby confirming that 1A8C3C10 supernate in factcontained IL-2. Moreover, the test results indicated that enhancement ofthe Con A stimulated thymocyte mitogenesis is related to the IL-2activity of the given condition medium since [³ H]Tdr incorporationproportionally decreased with increased dilution of all three differenttypes of condition media.

Confirmation of 1A8C3C10 Hybrid Origin

Through phenotypic characterization, the 1A8C3C10 hybrid clonal cellswere found to express both Thy 1.1 and Thy 1.2 differentiation antigens.The LBRM-33 cells are known to display the Thy 1.2 antigen but not theThy 1.1 antigen. Conversely, the BW 5147 cells are known to express theThy 1.1 antigen but not the Thy 1.2 antigen. Thus, the expression ofboth cell surface markers by the 1A8C3C10 hybrid T cell line confirmsthat this hybridoma cell was a product of the fusion of LBRM-33 cellswith BW 5147 tumor cells.

EXAMPLE 1

LBRM-33 murine leukemic tumor cells were stimulated for 12 hours with a1.0% by volume concentration of PHA (PHA, Grand Island BiochemicalCompany, Grand Island, NY) in a tissue culture medium composed of 20milliliters of RPMI 1640 medium supplemented with 5% by volume,heat-inactivated (56√ C. for thirty minutes) FCS. The culture wasmaintained at approximately 37° C. in a humidified atmosphere of 5%carbon dioxide in air. After PHA stimulation, the LBRM-33 lymphoma cellswere washed several times with a mixture composed of RPMI 1640 and 5% byvolume heat-inactivated FCS.

After the washing procedure, the LBRM-33 cells were fused with the BW5147 cells by mixing approximately 2×10⁷ of the PHA-stimulated LBRM-33cells with approximately 5×10⁶ drug-marked BW 5147 driver cells. Thecellular mixture was pelleted by centrifuging for five minutes at 160×g. One milliliter mixture of 40% (weight/volume) PEG in RPMI 1640 (pH7.2) was gradually added to the fusin pellet. The PEG treated cellmixture was then pelleted again by centrifuging for twelve minutes at300× g. Next, the pellet was resuspended by carefully adding, over a 5-7minute period, 10 milliliters of RPMI 1640 supplemented with 10% byvolume FCS. This cell solution was then pelleted a final time bycentrifuging for five minutes at 160× g.

Constitutive IL2 production by the hybridoma cells derived from thefusion of parent LBRM-33 cells and BW 5147 cells was achieved byculturing the cell pellet resulting from the last centrifugationprocedure, set forth in the above paragraph. The cell pellet wassuspended in 100 milliliters of Click's medium supplemented with 10% byvolume, heat-inactivated (56° C. for 30 minutes) FCS, 100 millimolar ofsodium pyruvate, 25 millimolar of Hepe's buffer, 16 millimolar ofNaHCO₃, 50 units per milliliter of penicillin, 50 micrograms permilliliter of streptomycin and 300 micrograms per milliliter of freshL-glutamine. Approximately 2×10⁸ BALB/c thymocyte cells were added tothe cell suspension to serve as filler cells. To prevent growth ofunfused BW 5147 driver cells, suppressing agents (HAT) composed of 13.6micrograms of hypoxanthine per milliliter of medium, 0.176 micrograms ofaminopterin per milliliter of culture and 3.88 micrograms of thymidineper milliliter of culture. The entire cell population and medium wasthen divided into individual 200 microliter aliquots in flat-bottomedmicroplate wells (No. 3596, Costar, Inc., Data Packaging, Cambridge,MA). The cultures were all maintained at approximately 37° C. in ahumidified atmosphere of 5% CO₂ in air. Periodically, the supernatesfrom the microculture wells that were found to contain hybrid cellgrowth were assayed for IL-2 activity using the microassay procedure asdiscussed above.

EXAMPLE 2

IL-2 was constitutively produced by culturing a concentration of 5×10⁵1A8C3C10 cells per milliliter in 25 cm² tissue culture flasks (#3013,Falcon Plastics, Oxnard, CA) in 5 milliliter aliquots of HAT-containingClick's medium. The medium was supplemented with 10% by volume,heat-inactivated (56° C. for 30 minutes) FCS, 100 millimolar of sodiumpyruvate, 25 millimolar of Hepe's buffer, 16 millimolar of NaHCO₃, 50units per milliliter of penicillin, 50 microgram per milliliterstreptomycin and 300 micrograms per milliliter of fresh L-glutamine. Theculture was maintained at approximately 37° C. in a humidifiedatmosphere of 5% CO₂ in air. Four days later, the supernate was found tocontain 19-20 units/milliliter of IL-2 activity.

As will be apparent to those skilled in the art to which the inventionis addressed, the present invention may be carried out by using hybrid Tcell lines, culture media, culture media additives, mitogen stimulants,drug-sensitizers, fusing agents and suppressing agents other than thosespecifically disclosed above, without departing from the spirit oressential characteristics of the invention. The particular processesdescribed above are therefore to be considered in all respects asillustrative and not restrictive, i.e. the scope of the presentinvention is as set forth in the appended claims rather than beinglimited to the examples of the IL-2 producing processes as set forth inthe foregoing description.

What is claimed is:
 1. A process for constitutively producing murineinterleukin-2, comprising:stimulating murine mononuclear leukocytes in afirst culture medium containing a T cell mitogen stimulant; markingmurine neoplastic cells with a sensitizing drug to produce driver cells;fusing said murine mononuclear leukocyte cells with said driver cells toproduce a hybridoma capable of constitutively producing interleukin-2;and culturing the hybridoma cells in a second culture medium, containingselective suppressing agents which allow only hybrid cells toproliferate.
 2. The process of claim 1, wherein the murine mononuclearleukocytes include malignant murine cells.
 3. The process of claim 2,wherein said malignant cells are T leukemic cells or T lymphoma cells.4. The process of claim 3, wherein said T lymphoma cells are LBRM-33cells.
 5. The process of claim 4, wherein said T lymphoma cells areclones of said LBRM-33 cells.
 6. The process of claim 3, wherein saidmalignant cells are clones of said T lymphoma cells.
 7. The process ofclaim 6, wherein said clones of T lymphoma cells are clones of LBRM-33lymphoma murine T cells.
 8. The process of claim 1, wherein said T cellmitogen in said first culture medium is a compound selected from thegroup consisting of phytohemagglutinin, concanavalin A, and pokeweedmitogen.
 9. The process of claim 8, wherein said first culture mediumincludes Roswell Park Memorial Institute medium
 1640. 10. The process ofclaim 8, wherein said first culture medium includes Click's medium orDulbecco Modified Eagle's medium.
 11. The process of claim 1, whereinsaid first culture medium is a compound selected from the groupconsisting of Roswell Park Memorial Institute medium 1640, Click'smedium and Dulbecco Modified Eagle's medium.
 12. The process of claim 2,wherein said T cell mitogen in said first culture medium is a compoundselected from the group consisting of phytohemagglutinin, concanavalin Aand pokeweed mitogen.
 13. The process of claim 12, wherein said firstculture medium includes Roswell Park Memorial Institute medium
 1640. 14.The process of claim 12, wherein said first culture medium includesClick's medium or Dulbecco Modified Eagle's medium.
 15. The process ofclaim 3, wherein said T cell mitogen in said first culture medium is acompound selected from the group consisting of phytohemagglutinin,concanavalin A and pokeweed mitogen.
 16. The process of claim 15,wherein said first culture medium includes Roswell Park MemorialInstitute medium
 1640. 17. The process of claim 15, wherein said firstculture medium includes Click's medium or Dulbecco Modified Eagle'smedium.
 18. The process of claim 1, wherein said murine driver cellsinclude drug marked, ouabain or 8-azaguanine resistant, malignant Tcells.
 19. The process of claim 18, wherein said driver cells includedrug marked BW 5147 thymocytes.
 20. The process of claim 18, whereinsaid sensitizing drug is a compound selected from the group consistingof ouabain, glutathione and 8-azaguanine.
 21. The process of claim 1,wherein the step of fusing the murine mononuclear leukocyte cells withdrug sensitive driver cells includes mixing the murine mononuclearleukocyte cells with the drug sensitive driver cells, and adding afusion compound selected from the group consisting of polyethyleneglycol and deoxyribonucleic acid transforming viruses.
 22. The processof claim 21, wherein said deoxyribonucleic acid transforming virusesinclude Sendai virus or the fusion protein obtained therefrom.
 23. Theprocess of claim 21, wherein said fusion compound further includes athird tissue culture medium.
 24. The process of claim 23, wherein saidthird tissue culture medium includes Roswell Park Memorial Institutemedium 1640, Click's medium or Dulbecco Modified Eagle's medium.
 25. Theprocess of claim 1, wherein said second culture medium includes acompound selected from a group consisting of Roswell Park MemorialInstitute medium 1640, Click's medium or Dulbecco Modified Eagle'smedium.
 26. The process of claim 25, wherein said second culture mediumcontains one or more compounds selected from the group consisting offetal calf serum, sodium pyruvate, Hepe's buffer, NaHCO₃, penicillin,streptomycin and L-glutamine.
 27. The process of claim 26, wherein saidsecond culture medium includes suppressing agents to preclude the growthof unfused driver cells.
 28. The process of claim 27, wherein saidsuppressing agents include one or more compounds selected from the groupconsisting of hypoxanthine, aminopterin and thymidine.
 29. The processof claim 25, wherein said second culture medium further includes feedercells.
 30. The process of claim 29, wherein said feeder cells includescells selected from the group consisting of murine thymocytes, murinespleen cells, irradiated murine peritoneal exudate cells and murinemacrophages.
 31. The process of claim 25, wherein said second culturemedium further includes feeder cells.
 32. The process of claim 1,further including the step of cloning said hybridoma cells.
 33. Theprocess of claim 32, wherein said hybridoma cells are cloned in a fourthculture medium.
 34. The process of claim 33, wherein said fourth culturemedium includes a compound selected from the group consisting of RoswellPark Memorial Institute medium 1640, Click's medium and DulbeccoModified Eagle's medium.
 35. The process of claim 34, wherein saidfourth culture medium contains one or more compounds selected from thegroup consisting of fetal calf serum, sodium pyruvate, Hepe's buffer,NaHCO₃, penicillin, streptomycin and L-glutamine.
 36. The process ofclaim 34, wherein said fourth culture medium further includes feedercells.
 37. The process of claim 36, wherein said feeder cells includecells selected from the group consisting of murine thymocytes, murinespleen cells, irradiated murine peritoneal exudate cells and murinemicrophages.
 38. A process for producing IL-2 from a hybrid murine Tcell line, comprising culturing in a tissue culture medium, hybrid Tcells capable of constitutively producing IL-2, said hybrid cellsproduced by fusing mitogen stimulated, malignant murine IL-2 producercells with drug marked murine driver cells.
 39. The process of claim 38,whereinsaid malignant murine IL-2 producer cells are T leukemic or Tlymphoma cells.
 40. The process of claim 39, wherein said T lymphomacells are LBRM-33 cells.
 41. The process of claim 40, wherein said Tlymphoma cells are clones of said LBRM-33 cells produced by separatingsaid LBRM-33 cell line into individual cells and then culturing theindividual cells in a culture medium.
 42. The process of claim 38,wherein said murine driver cells are T leukemic cells or T lymphomacells.
 43. The process of claim 42, wherein said murine lymphoma cellsare BW 5147 cells.
 44. The process of claim 38, wherein said hybrid Tcells are clones derived from parent hybrid T cells produced by fusionof murine, mitogen stimulated, IL-2 producer cells with drug-sensitivemurine driver cells.
 45. The process of claim 38, wherein said culturemedium includes a compound selected from the group consisting of RoswellPark Memorial Institute medium 1640, Click's medium, and DulbeccoModified Eagle's medium.
 46. The process of claim 45, wherein saidculture medium further includes driver cell suppressing agents toprevent proliferation of uncoupled driver cells.
 47. The process ofclaim 46, wherein said suppressing agents include hypoxanthine,aminopterin and thymidine.
 48. The process of claim 45, wherein saidculture medium further includes feeder cells.
 49. The process of claim48, wherein said feeder cells include cells selected from the groupconsisting of murine thymocytes, murine spleen cells, irradiated murineperitoneal exudate cells and murine macrophages.